dna is the genetic component of lifedmohnen/bcmb3100/lecturenoteschap33-15-2slides.pdfdna is the...

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BCMB 3100 - Nucleic Acids - Chapter 33

•Discovery of DNA

•Nucleotides, nucleosides & bases

•Polynucleotides

•DNA as genetic material

•Structure of double-stranded DNA

•Chromatin

•RNA

•Nucleases

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DNA is the genetic component of life

DNA RNA PROTEIN

Central Dogma for Biological Information Flow

Friedrich Miescher (1869): discovered DNA (nuclein nucleic acid

C, H, O, N, P

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DNA and RNA are made up of nucleotides

______________: base + sugar + phosphate(n)

deoxyribonucleotide (sugar = 2-deoxyribose)

ribonucleotide (sugar = ribose)

____________: base + sugar

__________ of nucleotides: heterocyclic rings containing nitrogen

Two class of bases: ____________ and ________

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Purines and Pyrimidines

See Fig. 33.5

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See Fig 33.5 Major pyrimidines and purines

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Tautomers of adenine and cytosine Amino versus Imino

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Tautomers of guanine, thymine and uracilLactam versus Lactim

Predominant forms

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Ribose and Deoxyribose

RNA DNA

See 33.3 Figure

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Nucleosides

See Fig. 33.6 Nucleoside structures

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Two conformations of nucleosides & nucleotides are possible due to rotation around the glycosidic bond:syn and anti

The _________conformation predominates

See Fig 33.6

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See Fig 33.7 Chemical structure of a __________

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Structures of the deoxyribonucleoside-5’-monophosphates

See Fig 33.7

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(continued)

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In vivo the negatively charged phosphates on nucleotides are

complexed with cations or positively charged proteins

***

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•Discovery of DNA


•Polynucleotides



•Chromatin

•RNA

•Nucleases

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A. Nucleotides joined by 3’-5’ phosphodiester linkages

Structure of the tetranucleotide

pdApdGpdTpdC

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Backbone of Nucleic Acids

Fig 33.4

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Story of DNA as Genetic Material

Discovery of the structure of double stranded DNA, 1953

James Watson, Francis Crick, Rosalind Franklin, Maurice Wilkins

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S = capsular

polysaccharide

= death

R= no capsule = live

Isolation & characterization

of the transforming

principle proved the chemical

make-up of the

genetic material

Evidence that

DNA is the

genetic

material in cells!!

(1)

(2)

(3)

Evidence for transforming

principle!

(4)21

Both set of phage were infective

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Nature (1953) 171:737

April

Nature (1953) 171:964

May

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Adjacent nucleotides

can hydrogen bond to

each other

see Fig 33.12

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•Discovery of DNA


•Polynucleotides



•Chromatin

•RNA

•Nucleases

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DNA is double-stranded with equal ratios of G:C and of A:T. However, the ratio of

(G+C):(A+T) varies in an species specific manner

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Complementary base

pairing and stacking in

DNA

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• Structure of B-DNA

• Sugar phosphate backbone outside

• Stacking creates two unequal grooves (major and minor)

• Hydrophobic attractionbetween the bases

• Van der Waals contact between bases

• H-bonds between bases

• Electrostatic repulsion between phosphates inhibited by cations (Mg++)

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Fig 33.13

Double helix emphasizing the charge

on the phosphate groups

Fig 33.11

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Watson and Crick discovered structure of

B-DNA.

Most common form of DNA

under physiological conditions.

B-DNA - Ball-and-stick model

B-DNA - Space-filling model

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Forms of DNA

AB

Z

Dehydrated DNADNA in vivo

in some GC rich regions

RNA:DNA hybridds RNA left-handed DNA

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Fig 33.17

B-DNA A-DNA

Dehydrated DNA

RNA:DNA hybridds RNA

DNA in vivo

Fig 33.17

Z-DNA

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Fig 33.20

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Fig 33.19

Major groove: wider - 12Å; deeper – 8.5Å

Minor groove: 6Å wide; 7.5Å deep

DNA molecules vary greatly in lengthdepending upon the organism and organelle

Species Length Genome size

E. coli 4.2 x 106 bp same

fruit fly 62 x 106 bp 130 x 106bp

mitochondria 0.015 x 106 bp same (from mammals; can be up to 2.5 x 108 in plants)(circular in mammals; can be linear or circular in plants)

Human 240 x 106 bp 3200 x 106 bp(46 chromosomes)

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Absorption spectra of double-stranded and single-stranded DNA

• Double-stranded(ds) DNA absorbance max 260 nm

• _____________ absorbs more than ds DNA

• dsDNA can be denatured by heat and chaotropic agents

• Extent of denaturation can be measured by OD260

How can you detect DNA in solution?

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Melting curve for DNA

Temperature at which amount of dsDNA = ssDNA is Tm(____________)

Tm for poly GCis greater than

Tm for poly AT

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•Discovery of DNA


•Polynucleotides



•RNA

•Chromatin

•Nucleases

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Stem-loop structures in RNA

• ssRNA can also have ds regions

• __________ or _______ can form from short regions of complementary base pairs

• Stem: base-paired nucleotides

• Loop: noncomplementary nucleotides

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Fig 33.30

Stem Loop Structures

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Four Classes of RNA in living organisms

______________ (rRNA) - ~80% of total RNA, part of ribosomes (translation machinery)

•____________ (tRNA) - ~15% of total RNA, 73-95 nucleotides long, carry activated amino acids to ribosomes during translation

______________ (mRNA) - linear “copies” of DNA that encode genetic information. Encode primary structure of protein. ~1-3% of total RNA, relatively unstable

___________ - may have catalytic activity and/or associate with proteins to enhance activity, some involved with RNA processing in the nucleus

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Alternative Classification of RNA

• RNAs involved in protein synthesisrRNA, tRNA, mRNA, others

• RNAs involved in post-transcriptional modification or DNA replication

modification or DNA replicationsnRNA, snoRNA, SmY, RNase P, others

• Regulatory RNAsaRNA (antisense RNA), miRNA (microRNA),

siRNA (small interfering RNA), others

• Parasitic RNAs

• Other RNAs

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•Discovery of DNA


•Polynucleotides



•RNA

•Chromatin

•Nucleases

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Structure of supercoiled DNA. Circular B-DNA has 10.4 bases/turn of helix. If DNA is underwound (or overwound), it is supercoiled to restore 10.4 bases/turn. Supercoiling is done

by topoisomerases.

See Fig. 33.22

Relaxed;(10.4 base pairs per turn

of the double helix)

Supercoiled (underwound)[supercoils are caused by underwinding or overwinding]

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Human topoisomerase I bound to DNA

• Topoisomerases can add or remove supercoils in DNA

• Cleave one or both DNA strands, unwind or overwind by rotating cleaved ends, then rejoin ends

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• In the nucleus DNA is found as ______________

• Chromatin: an association of DNA with proteins (mostly histones) compact & manageable packing. Chromatin looks like long threads of 30 nm diameter.

• Histones - the major proteins of chromatin

• Eukaryotes contain five small, basic histone proteins

containing many lysines and arginines:

H1, H2A, H2B, H3, and H4

• Positively charged histones bind to negatively-charged sugar-phosphates of DNA

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A structural unit in chromatin is the _____________

Nucleosome: a ~200 bp DNA strand wound around a histone core.

Chromatin treated with a low salt solution extends into a “beads on a string” structure. Beads are the nucleosomes; the string is DNA.

Electron micrograph of chromatin

Fig. 33.24

Electron micrograph of chromatin

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Histone octamer

Nucleosome core particle Fig. 33.26

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Nucleosome

146 bp

54 bp

Nucleosome gives 10-fold packing

Fig. 33.25

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Solenoid: a higher level of chromatin structure in which adjacaent nucleosome associate via histone H1

Solenoid give further4-fold packing

Histone-depleted chromosome scaffold. Attachment of DNA to RNA-protein scaffold gives further

200-fold packing

Protein scaffold Loops attached to scaffold

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Fig. 33.28

Representation of the compaction of DNA into a eukaryotic chromosome

Final chromosome is 1/8000 of length of B-DNA.

This allows DNA to be packaged into cells. For example, the largest human chromosome is 2.4 x 108 bp.

This chromosome would be 8.2 cm long if it were not packaged as chromatin(as opposed to 2-10 µm)!!

http://www.answers.com/topic/chromosome

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1685232/pdf/ajhg00205-0039.pdf

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•Discovery of DNA


•Polynucleotides



•RNA

•Chromatin

•Nucleases

Nucleases and Hydrolysis of Nucleic Acids

• Nucleases - hydrolyze phosphodiester bondsRNases (RNA substrates)DNases (DNA substrates)

• May cleave either the 3’- or the 5’- ester bond of a 3’-5’ phosphodiester linkage

• Exonucleases start at the end of a chain

• Endonucleases hydrolyze sites within a chain

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• Nuclease cleavage sites

• Cleavage at bond A generates a 5’-phosphate and a 3’ OH terminus

• Cleavage at bond B generates a 3’-phosphate and a 5’-hydroxyl terminus

A = cleavage of 3’- ester bond

B = cleavage of 5’- ester bond

Cleavage of 3’ ester of Guanylate

5’ ….pGpCpAp…3’ + H2O 5’….pG + pCpAp… 3’

Cleavage of 5’ ester of Guanylate

5’ ….pGpCpAp…3’ + H2O 5’….p +GpCpAp… 3’

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DNA is stable in basic solution

RNA is unstable in base

Alkaline Hydrolysis of RNA

RNA is less stable than DNA because RNA is sensitive to hydrolysis in basic solutions

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(From previous page)

Ribonuclease-Catalyzed Hydrolysis of RNARNase A cleaves 5’ ester to right of pyrimidines

5’…pApGpCpAp…3’ → 5’…pApGpCp + Ap…3’

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Enzyme-catalyzed cleavage by RNAse A results, specifically, in a 3’-phosphate product

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_____________________: site-specific endodeoxyribonucleases causing cleavage of both strands of DNA at points within or near the specific site recognized by the enzymes; important tools in genetic engineering

______________________: catalyze both methylation of host DNA and cleavage of non-methylated DNA at recognition site

______________________: cleave non-methylated DNA at recognition site

Most restriction enzymes recognize palindromes: inverted sequences with two-fold symmetry over two strands

5’AAGAATTCGG3’

3’ATCTTAAGCC5’

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• Methylation and restriction at the EcoR1 site

EcoRI

GAATTCCTTAAG

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EcoRI

GAATTCCTTAAG

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D. EcoR1 Binds Tightly to DNA

• EcoR1 has 2 identical subunits (purple and yellow)

• Bound to a fragment of DNA (strands blue and green)

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Uses of Restriction Endonucleases

• Developing restriction maps (indicates specific cleavage sites in a DNA fragment)

• Map of bacteriophage showing cleavage sites of some restriction enzymes

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• Restriction digest of bacteriophage

• Four restriction enzymes used

• Sizing gel separates fragments (smallest move fastest)

top

bottom

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• DNA Fingerprinting

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